1. Field of the Invention
The field of the invention relates to plants and plant genes, including both plant mutants and transgenic plants containing a gene that confers an ethylene insensitive phenotype. Also encompassed by the invention are methods of using the disclosed plant gene to confer an ethylene insensitive phenotype.
2. Description of the Related Art
The plant hormone ethylene plays a central role in a variety of processes, including wounding, pathogen attack, cell fate and elongation, senescence, and fruit ripening. The classical triple response to ethylene, inhibited hypocotyl and root elongation, thickening of the hypocotyl, and exaggeration of the apical hook, has been used to study the signaling pathway that mediates this hormone response. Molecular genetic studies in Arabidopsis thaliana have exploited the triple response to ethylene, and resulted in the identification of mutants defective in this response. Epistasis studies with plants that are insensitive to the hormone or display a constitutive response to ethylene have suggested a signaling pathway in which one group of proteins acts to antagonize pathway activity in the absence of the hormone, and a second group functions to transmit the signal and initiate physiological responses.
The ETR1 gene and a family of related genes, including EIN4, ETR2, ERS1, and ERS2, encode ethylene receptors. Ethylene binding is mediated by the hydrophobic amino terminus of ETR1 in a copper dependent manner. Point mutations in this domain can disrupt ethylene binding, and result in dominant ethylene insensitivity, suggesting that the proteins actively suppress pathway activity in the absence of the hormone, and that ethylene binding removes this inhibitory activity. Genetic experiments in which combining loss of function alleles of at least three family members resulted in constitutive pathway activity in the absence of ethylene confirmed the idea that the receptors negatively regulate ethylene signaling in the absence of the hormone. The important role copper plays in receptor function was underscored by the identification of the RAN1 gene, which bears significant sequence similarity to the Menkes/Wilson disease copper transporter. Disruption of RAN1 by mutation or co-suppression resulted in plants that display a constitutive triple response, suggesting that copper is required for the nominal formation or proper structure of a receptor complex in addition to ethylene binding.
Mutation of the Raf kinase homolog CTR1 also results in a constitutive triple response in the absence of ethylene. Based on epistasis studies, CTR1 functions downstream of the ethylene receptors, and represses signaling pathway activity in the absence of the hormone. The repressive roles played by both CTR1 and the receptors suggests a functional link between the two. Indeed, protein interaction studies have shown that CTR1 and ETR1 physically interact, but the manner by which ETR1 may activate CTR1 is unknown. Although the homology of CTR1 to Raf suggests a downstream MAP kinase signaling cascade, the manner by which CTR1 represses ethylene signaling is also unknown.
Mutations of the EIN2 gene are recessive and also confer complete ethylene insensitivity. Epistasis testing indicates that EIN2 functions downstream of CTR1, and consequently is thought to play a central role in ethylene responses. EIN2 possesses an amino terminus that is predicted to contain multiple transmembrane domains, and is similar to metal transporters of the Nramp family. The exact manner in which EIN2 functions to activate the ethylene signaling pathway is unclear, however it has been shown that expression of its carboxyl end is sufficient to stimulate ethylene-dependent effects, including the triple response.
Terminal components of the primary ethylene signaling pathway are encoded by the EIN3 gene and a related family of EIL genes. ein3 mutations partially block activation by both ctr1 mutations and the EIN2 carboxyl end, indicating that it functions downstream of EIN2 in the signaling cascade. EIN3 and EIL proteins represent a novel class of transcription factors, and have been found to bind as dimers to a unique site found in some promoters of a second class of transcription factors termed ethylene response element binding proteins (EPEBP). Many of these factors bind to the GCC box found in many ethylene responsive genes that are activated several hours after ethylene treatment. An EIN3 binding site was found in the promoter of one such factor, ERF1, which was activated within fifteen minutes of ethylene treatment in an EIN3-dependent manner. Overexpression of ERF1 activated a subset of ethylene responses and bypassed an ein3 mutation, indicating that it functions downstream of EIN3 in the signaling cascade. These data indicate that a complete ethylene response is mediated by a series of transcription factors that act in sequential steps. EIN3 and EIL proteins act very early in the transcriptional cascade, whereas ERF1 and related proteins act later.
Roman et al. described a mutant plant, ein6 (Roman, et al. (1995) “Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway” Genetics 139(3): 1393–1409) This mutant plant demonstrated ethylene insensitivity, which was assumed to be the result of a single recessive mutation. The EIN6 gene was not identified. Unlike etr1 and ein2 mutants, ein6 mutant plants were found to retain some sensitivity to ethylene, with the root being less sensitive to ethylene than the hypocotyl. However, the original work mischaracterized the ein6 mutant plant as the phenotype was thought to be due to a single mutation.
In addition, the chromosomal location and DNA/Protein sequence of the EIN6 gene was not described, thus hindering in-depth studies of this mutation. However, as described below, the present application describes the isolation and characterization of the EIN6 gene, as well as the mutant allele, ein6.